1. Field of the Invention
The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to molecular cloning and characterization of homologous 28-kDa protein genes in Ehrlichia canis, a multigene locus encoding the 28-kDa homologous proteins of Ehrlichia canis and uses thereof.
2. Description of the Related Art
Canine ehrlichiosis, also known as canine tropical pancytopenia, is a tick-borne rickettsial disease of dogs first described in Africa in 1935 and the United States in 1963 (Donatien and Lestoquard, 1935; Ewing, 1963). The disease became better recognized after an epizootic outbreak occurred in United States military dogs during the Vietnam War (Walker et al., 1970)
The etiologic agent of canine ehrlichiosis is Ehrlichia canis, a small, gram-negative, obligate intracellular bacterium which exhibits tropism for mononuclear phagocytes (Nyindo et al., 1971) and is transmitted by the brown dog tick, Rhipicephalus sanguineus (Groves et al., 1975). The progression of canine ehrlichiosis occurs in three phases, acute, subclinical and chronic. The acute phase is characterized by fever, anorexia, depression, lymphadenopathy and mild thrombocytopenia (Troy and Forrester, 1990). Dogs typically recover from the acute phase, but become persistently infected carriers of the organism without clinical signs of disease for months or even years (Harrus et al., 1998). A chronic phase develops in some cases that is characterized by thrombocytopenia, hyperglobulinemia, anorexia, emaciation, and hemorrhage, particularly epistaxis, followed by death (Troy and Forrester, 1990).
Regulation of surface antigenicity may be an important mechanism for the establishment of such persistent infections in the host. Although disease pathogenesis is poorly understood, multigene families described in members of the related genera Ehrlichia, Anaplasma, and Cowdria may be involved in variation of major surface antigen expression thereby evading immune surveillance. Anaplasma marginale, an organism closely related to E. canis, exhibits variation of major surface protein 3 (msp-3) genes resulting in antigenic polymorphism among strains (Alleman et al., 1997).
Molecular taxonomic analysis based on the 16S rRNA gene has determined that E. canis and E. chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME), are closely related (Anderson et al., 1991; Anderson et al., 1992; Dawson et al., 1991; Chen et al., 1994). Considerable cross reactivity of the 64, 47, 40, 30, 29 and 23-kDa antigens between E. canis and E. chaffeensis has been reported (Chen et al., 1994; Chen et al., 1997; Rikihisa et al., 1994; Rikihisa et al., 1992). Analysis of immunoreactive antigens with human and canine convalescent phase sera by immunoblot has resulted in the identification of numerous immunodominant proteins of E. canis, including a 30-kDa protein (Chen et al., 1997). In addition, a 30-kDa protein of E. canis has been described as a major immunodominant antigen recognized early in the immune response that is antigenically distinct from the 30-kDa protein of E. chaffeensis (Rikihisa et al., 1992; Rikihisa et al., 1994). Other immunodominant proteins of E. canis with molecular masses ranging from 20 to 30-kDa have also been identified (Brouqui et al., 1992; Nyindo et al., 1991; Chen et al., 1994; Chen et al., 1997).
Homologous 28-32 kDa immunodominant proteins encoded by multigene families have been reported in related organisms including, E. chaffeensis and Cowdria ruminantium (Sulsona et al., 1999; Ohashi et al., 1998a; Reddy et al., 1998). Recently, characterization of a 21 member multigene family encoding proteins of 23 to 28-kDa has been described in E. chaffeensis (Yu et al., 2000). The E. chaffeensis 28-kDa outer membrane proteins are surface exposed, and contain three major hypervariable regions (Ohashi et al., 1998a). The recombinant E. chaffeensis P28 appeared to provide protection against homologous challenge infection in mice, and antisera produced against the recombinant protein cross reacted with a 30-kDa protein of E. canis (Ohashi et al., 1998a). Diversity in the p28 gene among E. chaffeensis isolates has been reported (Yu et al., 1999a), and studies using monoclonal antibodies have further demonstrated diversity in the expressed P28 proteins (Yu et al., 1993). Conversely, complete conservation of a p28 genes in geographically different isolates of E. canis has been reported and suggests that E. canis may be conserved in North America (McBride et al., 1999, 2000).
The prior art is deficient in the lack of cloning and characterization of new homologous 28-kDa immunoreactive protein genes of Ehrlichia canis and a single multigene locus containing the homologous 28-kDa protein genes. Further, The prior art is deficient in the lack of recombinant proteins of such immunoreactive genes of Ehrlichia canis. The present invention fulfills this long-standing need and desire in the art.